The family of a US government scientist who fell to his death from a New York hotel window six decades ago have launched a lawsuit for damages against the CIA, alleging the agency was involved in his murder and a subsequent cover-up.

In one of the most notorious cases in the organisation's history, bioweapons expert Frank Olson died in 1953, nine days after he was given LSD by agency officials without his knowledge.

In the lawsuit, filed in the US district court in Washington on Wednesday, Olson's sons Eric and Nils claim their father was murdered after he witnessed extreme interrogations in which the CIA killed suspects using the biological agents he had developed.

The CIA has long denied any foul play, though it was forced to admit in 1975, more than 20 years after the death, that the scientist had been given LSD in a spiked glass of Cointreau. The agency, which originally told the family the death was a result of job-induced stress, has since maintained that it was a drug-induced suicide.

But in a statement on Wednesday, Eric Olson said: "The evidence shows that our father was killed in their custody. They have lied to us ever since, withholding documents and information, and changing their story when convenient.

"We were just little boys and they took away our lives – the CIA didn't kill only our father, they killed our entire family again and again and again."

The lawsuit alleges that even when the drug details emerged, the CIA embarked on a "multi-decade cover-up that continues to this day."

Olson began work at the special operations division (SOP) of the army's biological laboratory at Fort Detrick in Maryland in 1950. The CIA worked with the SOP researching biological agents and chemical weapons. In 1952 and 1953, he was focused on bioweapons that could be transmitted through the air, according to the lawsuit.

In the year of his death, Olson visited Porton Down, the UK's biological and chemical warfare research centre in Wiltshire, as well as bases in Paris, Norway, and West Germany. During these trips, according to the family's lawsuit, he "witnessed extreme interrogations in which the CIA committed murder using biological agents that Dr Olson had developed".

The lawsuit gives no details of the deaths or where they occurred.

The family said Olson was disturbed by what he had seen and told his wife, Alice, he wanted to quit.

On 19 November 1953 he was taken to a secret meeting Deep Creek Lake, Maryland, where he was given the drink laced with LSD. On 24 November, according to the lawsuit, he told a colleague he wanted to resign.

But instead, on Thanksgiving weekend, he travelled to New York for a psychiatric evaluation and checked into the Statler Hotel. In the early hours of 28 November, he crashed through the window of the 13th-floor room he was sharing with a CIA doctor and plunged to his death in the street below.

The family lawsuit alleges that, immediately following his death, a person in Olson's room made a phone call. The hotel operator overheard one party say "Well, he's gone." The person on the other end responded simply "That's too bad."

The role of LSD in the death only emerged in 1975 during a series of post-Watergate era disclosures about CIA abuses, which revealed programmes on brainwashing, mind control and other human experiments during the early days of the cold war. The Olson case became a symbol for reckless CIA behaviour and government secrecy.

Soon after the revelations, Gerald Ford apologised to the family for an experiment gone wrong, the CIA promised a "complete file" of documents into his death and they were awarded a financial settlement.

But his sons, who have spent much of their adult lives searching for answers in the case, say their questions have been met with cover-ups and lies ever since. Eric Olson said the CIA had refused to provide documents to the family as recently as last year.

Over the years, the Olson family has uncovered evidence they believe supports their theory. Olson's body was exhumed in 1993 and a forensic scientist, James Starrs, concluded that he had probably been struck on the head and then thrown out of the window. Later, the New York district attorney conducted an investigation into his death which was inconclusive.

Scott Gilbert, lead counsel in the lawsuit, said: "It's unfathomable that our own government could stand by as its agents, operating on United States soil, killed an American citizen in cold blood, destroyed his family, and then allowed those directly responsible to walk away without so much as a blemish on their personnel files. Instead of putting its energy and resources into doing what is right, the United States – including this administration – has sought to bury this and hide the truth."

Jennifer Youngblood, a spokeswoman for the CIA, said the agency did not normally comment on pending court cases. However, she added: "Without commenting on this specific legal matter, CIA activities related to MK-Ultra [a behavioral engineering programme] have been thoroughly investigated over the years, and the agency co-operated with each of those investigations. MK-Ultra was investigated in 1975 by the Rockefeller commission and the Church committee, and in 1977 by the Senate select committee on intelligence and the Senate subcommittee on health and scientific research. In addition, tens of thousands of pages related to the programme have been declassified and released to the public."